As processes for producing nucleosides by reacting a saccharide residue donor such as nucleoside and ribose 1-phosphate enzymatically with a base donor, for example, there have been reported various processes for producing various purine nucleosides (Japanese Patent Publications Nos. 24475/1968, 28954/1968, 28955/1968, 28956/1968, 11116/1970, 14957/1973, Japanese Laid-Open Patent Publications Nos. 71495/1980, 18599/1981, 142293/1981, 164793/1981, 166199/1981, 63393/1983, 94396/1983, 170493/1993, etc.), processes for producing various pyrimidine nucleosides (Japanese Patent Publication No. 16478/1960, Japanese Laid-Open Patent Publications Nos. 102794/1981, 213397/1984, 239495/1985, etc.), and processes for producing other various nucleosides (Japanese Laid-Open Patent Publications Nos. 29720/1975, 146593/1982, 190396/1983, 216696/1983, 143599/1984, 179094/1984, 213397/1984, 120981/1985, 133896/1985, 31093/1988, 177797/1988, etc.).
However, although these enzymatic processes for production of nucleosides are excellent as compared with chemical synthetic methods with respect to substrate specificity and steric selectivity inherent in enzymatic reaction, the activity of the enzyme has not been sufficient, and they have not been found satisfactory with respect to yield in all cases.
Also, when the reaction is carried out at room temperature, lowering in yield which may be considered to be caused by contamination with bacteria is observed, and when the reaction is carried out at a higher temperature (e.g., 45.degree. C. or higher) in order to avoid contamination, the enzyme gradually becomes deactivated, consequently leading to a marked lowering in yield.
Generally speaking, synthesis of a compound will be brought about by inclination of the equilibrium between the formation reaction and the decomposition reaction toward the formation reaction. For this reason, for increasing the yield of the compound, it is important to promote the formation reaction and suppress the decomposition reaction, and this principle is not exceptional in the enzymatic process for the production of a compound.
Also, if the reaction temperature is made higher, the reaction rate will become more rapid, whereby the reaction will be completed within a shorter time and the solubility of the substrate will also be enhanced, and hence it has the possibility of producing the objective product with good yield.
When producing nucleosides by the use of nucleoside phosphorylase, for promoting the formation reaction of nucleosides, consideration must be taken from the two points of the activity of the enzyme itself to be used as a catalyst and the reaction conditions. Selection of the reaction conditions is no more than an auxiliary means for inducing the activity of the enzyme employed, and the drastic method for promoting the formation reaction to increase the yield of the objective compounds is to use nucleoside phosphorylase having excellent activity.
The nucleoside phosphorylases of the prior art which have been used in the production of nucleosides are for the most part those prepared from microorganisms which can be easily cultured. However, when the activity of the enzyme is examined from the efficiency of the reaction, specific activity, heat resistance, yield of the objective compound, etc., those which have been used in the prior art have not always been satisfactory.
On the other hand, concerning the enzyme which has been considered to participate in the decomposition reaction of the production of nucleosides, for example, nucleosidase, the method of inhibiting the nucleosidase by means of the immobilization method using a photocurable resin has been reported (Japanese Laid-Open Patent Publication No. 253393/1987). This method is excellent, but when using microorganism cells as an enzyme preparation, some microorganisms cannot be easily immobilized, thus the method lacks general purpose applicability.
The present inventors have screened various microorganisms in order to discover enzymes of excellent activity which can be used for enzymatic production of nucleosides and consequently discovered a group of microorganisms containing a large amount of heat-resistant nucleoside phosphorylase having extremely high specific activity, and having high nucleoside phosphorylase activity per unit cell weight among thermophiles belonging to the genus Bacillus.
In the prior art, nucleoside phosphorylase has been isolated and purified from Bacillus stearothermophilus which is a thermophile belonging to the genus Bacillus and the enzymatic properties of the enzyme have been reported (see J. Biol. Chem., 244, 3691-3697 (1969), Agric. Biol. Chem., 53, 2205-2210 (Aug. 23, 1989), Agric. Biol. Chem., 53, 3219-3224 (Dec. 23, 1989)). Also, a process for producing 5-methyluridine or thymidine by using microorganism cells of Bacillus stearothermophilus as an enzyme source has been reported (see Japanese Laid-Open Patent Publication No. 320995/1989 (published on Dec. 27, 1989), Agric. Biol. Chem., 53, 197-202 (Jan. 23, 1989)). However, the nucleoside phosphorylases of the above-mentioned reports, although having the advantage of heat resistance, have low specific activity and also low enzyme activity per unit cell weight and thus could not solve the problem of the prior art that no nucleoside can be efficiently produced. More specifically, when the yield of the nucleoside disclosed in Japanese Laid-Open Patent Publication No. 320995/1989 is represented in terms of its proportion relative to the base donor employed, it is at most around 30% (even if the reaction has occurred ideally, the yield of the objective product determined from the equilibrium constant of the enzyme reaction is 53 to 56%).